Method of treating or preventing oxidative stress-related disease
a technology of oxidative stress and disease, applied in the direction of biocide, anti-noxious agents, drug compositions, etc., can solve the problems of cell damage and death, achieve the effects of improving functional outcome, reducing brain damage, and improving functional outcom
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example 1
[0053]This example demonstrates a synthesis of a uric acid analog of formula (I) in an embodiment of the invention.
[0054]Commercially available derivatives are obtained from Sigma-Aldrich (St. Louis, Mo.). The methods used for the synthesis of methyl- and sulfur-derivatives of uric acid are similar to those described previously (Beaman et al., J. Org. Chem., 27, 986-990 (1962) and Maruyama et al., Nucleosides, Nucleotides, Nucleic Acids, 19, 1193-1203 (2000)) and structures are confirmed by 13C nuclear magnetic resonance (NMR) and ultra-violet (UV) spectral analyses. Uric acid analogs are assayed by high performance liquid chromatography (HPLC) and found to be of >97% purity. The solubilities of uric acid and compounds of formula (I) in cold water (mg / ml) are measured and set forth in Table 1.
TABLE 1WaterSolubilityCompound(mg / ml)uric acid0.1(comparative)mUA11mUA22mUA32sUA12sUA22sUA33sUA43
[0055]Thus, the water solubilities of the compounds of formula (I) are 10- to 30-fold greater th...
example 2
[0056]This example demonstrates a method to determine the pharmacokinetics of compounds of formula (I) in an embodiment of the invention.
[0057]Mice are administered intravenously 10 mg / kg (5.95 mmols) of uric acid as a reference compound and equimolar doses of its analogs (sUA4-2,6,8 trithio uric acid, 12.9 mg / kg; mUA2-1,7 dimethyl uric acid, 11.7 mg / kg; sUA2-6,8 dithio uric acid, 11.9 mg / kg). Three month-old male C57BL / 6 mice are injected with a compound and are euthanized 15, 30, 60 or 120 min later (1 mouse / time point / compound). Blood and brain tissues are rapidly removed, frozen on dry ice and kept at −80° C. before quantification of uric acid analogs. Plasma and cerebral cortical tissues are extracted in 10 volumes of 0.5 M perchloric acid and 1 mM ethylenediamine tetraacetic acid (EDTA). Levels of uric acid analogs in the extracts are quantified by HPLC methods employing a 25 cm RP C18 column, 25 mM KH2PO4 buffer (pH=4.6) with 1 ml / min flow rate and UV diode array detection.
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example 3
[0059]This example demonstrates a method of synaptosome preparation and an assay to determine antioxidant activity in an embodiment of the invention.
[0060]Synaptosomes are prepared from the cerebral cortex of 2-3 month-old adult male Sprague-Dawley rats and are cryopreserved using methods described previously (Begley et al., Brain Res. Protoc., 3, 76-82 (1998)). After thawing, cryopreserved synaptosomes are washed twice with Locke's buffer and plated in 24-well plates at a protein concentration of 250 μg per well. After a 30 min period to permit attachment of the synaptosomes to the substrate, synaptomoses are incubated for 1 h with compounds of formula (I) (1-10 μM), and then exposed for 4 h to freshly prepared 50 μM Fe2+ (FeSO4). After an additional 30 min incubation in the presence of 10 μM dihydrorhodamine 123 (DHR), a probe for ROS which fluoresces when oxidized (Keller et al., J. Neurochem., 69, 273-284 (1997)), the synaptosomes are washed twice with Locke's buffer and levels ...
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